Decreased Semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis
© Takagawa et al.; licensee BioMed Central Ltd. 2013
Received: 10 September 2012
Accepted: 12 January 2013
Published: 23 January 2013
Rheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not fully understood. Semaphorin3A (Sema3A) has an immune regulatory role. Neuropilin1 (NRP1), the primary receptor for Sema3A, is also a receptor for vascular endothelial growth factor 165 (VEGF165). It has been shown that Sema3A competitively antagonizes VEGF165 signaling. This study investigated whether Sema3A is expressed in synovial tissues, and is associated with disease activity and the histological features of synovial tissues from RA patients.
Human synovial tissues samples were obtained from RA and osteoarthritis (OA) patients. Disease activity of RA patients was calculated using the 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP). The histological features of RA synovial tissues were evaluated using Rooney’s inflammation scoring system. The localization of Sema3A, VEGF165 and NRP1 positive cells was immunohistochemically determined in synovial tissues. Expression levels of Sema3A, VEGF-A and NRP1 mRNA were determined using quantitative real-time polymerase chain reaction (qPCR).
In OA specimens, Sema3A, VEGF165 and NRP1 proteins were expressed in the synovial lining and inflammatory cells beneath the lining. Immunohistochemistry revealed the protein expression of Sema3A in synovial lining cells was decreased in RA tissues compared with OA samples. qPCR analysis demonstrated a significant reduction of Sema3A mRNA levels in RA synovial tissue samples than in OA and a significant correlation of the ratio of Sema3A/VEGF-A mRNA expression levels with DAS28-CRP (R = −0.449, p = 0.013). Sema3A mRNA levels also correlated with Rooney’s inflammation score, especially in perivascular infiltrates of lymphocytes (R = −0.506, p = 0.004), focal aggregates of lymphocytes (R = −0.501, p = 0.005) and diffuse infiltrates of lymphocytes (R = −0.536, p = 0.002).
Reduction of Sema3A expression in RA synovial tissues may contribute to pathogenesis of RA.
KeywordsRheumatoid arthritis Semaphorin3A Disease activity score Histological scoring
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction that accompanies the proliferation of synovial cells and blood vessels as well as invasion of inflammatory cells [1–3]. Although the initiating factors of RA are unknown, autoimmune reactions are activated in connective tissues. In RA joints, immune cells such as T and B cells invade the hyperplastic synovial membranes . Activated synovial T and B cells secrete various types of pro-inflammatory cytokines including interleukin-1 (IL-1), IL-17 and tumor necrosis factor-α (TNF-α). These cytokines induce the synthesis of matrix degrading enzymes in chondrocytes. Synovial fibroblasts also produce matrix-degrading enzymes and can invade cartilage, leading to its destruction [3, 4].
Semaphorins are a large family of proteins that function as guidance cues for axonal/dendritic projections. Class 3 semaphorins are vertebrate secreted proteins and include seven members, semaphorin3A (Sema3A) to Semaphorin3G (Sema3G) . Sema3A is a repulsive factor for sensory fibers, and Semaphorin3C (Sema3C) and Semaphorin3F (Sema3F) repulse sympathetic nerve fibers . Whereas the primary receptor for Sema3A is neuropilin1 (NRP1), Sema3F has a higher binding affinity to neuropilin2 (NRP2). Sema3C binds to both NRP1 and NRP2 [7, 8].
The action of Sema3A is not limited to the nervous system as NRP1 is expressed on endothelial cells, keratinocytes, T cells, and tumor cells in breast and prostate cancer. Sema3A inhibits angiogenesis, migration of keratinocytes, proliferation of T cells, and migration of tumor cells [8–11]. In addition, it was recently shown that Sema3A is involved in the entry of dendritic cells to the lymphatic system . Several studies have indicated that a reduction of Sema3A expression is involved in the exacerbation of autoimmune diseases, such as RA and systemic lupus erythematosus (SLE) [13, 14].
NRP1 mediates signal transduction through PlexinA co-receptors , which are classified into four sub-families, PlexinA1–4 . The Sema3A/NRP1/PlexinA complex regulates the actin cytoskeleton through small G-proteins, including Rac and Rho . In immune cells, the Rac family is associated with the proliferation and activation of B cells , and the activation of T cells induced by dendritic cells . NRP1 is also a putative marker of regulatory T cells , and therefore Sema3A/NRP1/PlexinA signaling may modulate regulatory T cell functions.
Vascular endothelial growth factor165 (VEGF165), a spliced isoform of VEGF-A , binds to NRP1 . VEGF is a key regulator of angiogenesis and is involved in the development of inflammation . In RA patients, serum VEGF levels positively correlate with disease activity score and joint destruction [24, 25]. Because NRP1 is a common receptor for Sema3A and VEGF165 [11, 26], the efficacy of VEGF165 is attributed to Sema3A expression. Indeed, the imbalance between Sema3A and VEGF expression levels is also associated with disease activity in several tumors [27–29].
These facts prompted us to investigate the possibility that Sema3A expression and/or the balance of Sema3A and VEGF165 expression may regulate the disease activity of RA including inflammation, angiogenesis and proliferation of synovial cells. We found that Sema3A expression was decreased in RA synovial tissues when compared with osteoarthritis (OA) samples. The Sema3A expression level was also significantly associated with the RA pathological score and disease activity score.
Patients and samples
Characteristics of patients with OA and RA
OA (n = 23)
RA (n = 30)
Age, mean ± SD (range, years)
76.6 ± 5.8 (64–84)
69.4 ± 8.1 (55–83)
Disease duration, mean ± SD (years)
10.7 ± 5.1
16.3 ± 9.1
CRP, mean ± SD (mg/dl)
0.16 ± 0.30
1.43 ± 1.21
DAS28-CRP, mean ± SD
3.74 ± 0.84
Prednisolone, n (%)
Methotrexate, n (%)
Sulfasalazine, n (%)
Bucillamine, n (%)
Cyclosporine, n (%)
Actarit, n (%)
TNF-α blockade, n (%)
Hematoxylin and eosin (HE) staining for histological assessment
Rooney’s inflammation scoring system for patients with rheumatoid arthritis
Proliferating blood vesselsc
Perivascular infiltrates of lymphocytesd
Focal aggregates of lymphocytese
Diffuse infiltrates of lymphocytesf
For immunostaining of Sema3A, antigen retrieval was performed by incubating at 95°C for 30 min in DAKO Target Retrieval Solution (pH 9.0; DAKO, Glostrup, Denmark). For immunostaining of NRP1, VEGF165, CD3 and CD20, the tissue sections were subjected to antigen retrieval by autoclaving in 10 mM citrate buffer (pH 6.0) for 15 min at 121°C. Slides were then treated with 0.3% H2O2 for 30 min to block endogenous peroxidases. Sections for Sema3A, NRP1 and VEGF165 staining were blocked with 10% normal goat serum. All sections were incubated at 4°C overnight with rabbit anti-Sema3A polyclonal antibody (1:200; Abcam, Cambridge, UK), mouse anti-CD3 monoclonal antibody (1:50; DAKO), mouse anti-CD20 monoclonal antibody (DAKO), rabbit anti-NRP1 polyclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-VEGF165 polyclonal antibody (1:100; Millipore, MA, USA). This was followed by incubation with Envision™, Rabbit/HRP (DAKO) for Sema3A, NRP1 and VEGF165 or Envision™/HRP (DAKO) for CD3 and CD20. Immunoreactivity was visualized using 3,3’-diaminobenzidine plus (DAB+, DAKO) for Sema3A, NRP1 and VEGF165, or 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, St. Louis, MO, USA) for CD3 and CD20. Finally, the sections were counterstained with hematoxylin and mounted. Controls for Sema3A immunohistochemistry included preabsorption and co-incubation of the antibody with the antigen peptide (1 μl/ml; Abcam).
Quantification of Sema3A immunostaining
Tissue sections immunostained with anti-Sema3A antibody were analyzed for 12 patients with OA and 12 patients with RA. The total immunostaining intensity in the lining layer was measured using a BZ-9000 microscope (Keyence, Osaka, Japan) equipped with Dynamic Cell count software BZ-H1C (Keyence). Immunostaining intensity per unit was calculated as described previously .
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from synovial tissues using an Illustra RNA spin Mini Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. RNA was reversed transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara Bio, Ohtsu, Japan). The cDNA synthesized from 1 μg of total RNA was used as the template in each reaction. qPCR analysis was performed using an Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems LLC) based on the TaqMan® PCR manufacturer’s protocol. The assay was performed in triplicate in optical 96-well reaction plates covered with optical adhesive cover in a volume of 10 μl containing 0.5 μl Taqman Gene Expression Assay 20X for human Sema3A (assay ID Hs01085496_m1, GenBank accession number NM_006080, Applied Biosystems LLC), VEGF-A (assay ID Hs00173626_m1, GenBank accession number NG_008732, Applied Biosystems LLC), NRP1 (assay ID Hs00826128_m1, GenBank accession number NM_003873; Applied Biosystems LLC) and β-actin (assay ID 4326315E, GenBank accession number NM_001101; Applied Biosystems LLC), 5 μl Taqman Fast Advanced Master Mix 2X, 2 μl cDNA template and 2.5 μl RNase-free water. The default ABI 7900HT amplification conditions were 20 sec at 95°C, followed by 1 sec at 95°C and 20 sec at 60°C for 40 cycles. A standard curve, derived from known serial dilutions of RA synovial tissue, was constructed to calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies. The obtained data were standardized using the reference gene, β-actin.
Synovial tissue specimens embedded in Optimum Cutting Temperature compound (Sakura Finetek Japan, Tokyo, Japan) were sectioned (5 μm thick). The sections were then fixed in cold acetone for 5 min at 4°C and rinsed in phosphate buffered saline (PBS). To eliminate nonspecific protein binding, the samples were incubated with 10% normal goat serum for 30 min at room temperature. The samples were incubated with rabbit anti-NRP1 polyclonal antibody (1:100; Santa Cruz Biotechnology) and mouse anti-CD20 monoclonal antibody (DAKO) overnight at 4°C. This was followed by incubation with Alexa 488-labeled goat anti-rabbit antibody (Applied Biosystems LLC, Foster City, CA, USA) and Alexa 594-labeled goat anti-mouse antibody (Applied Biosystems LLC) for 40 min at 37°C. Finally, the sections were mounted with aqueous mounting medium. The distributions were analyzed by confocal microscopy using a Zeiss LSM510 confocal laser microscope (Carl Zeiss, Oberkochen, Germany).
Statistical analyses were performed using SPSS 11.0 for Windows (SPSS Inc, Chicago, IL). The Mann–Whitney U-test and Spearman’s rank correlation coefficient were used to test the differences. A p value < 0.05 was considered significant. We calculated a posterior power of this study using G*Power (Faul, Erdfelder, Lang, & Buchner, 2007). All statistical powers in this study were greater than 80%.
Sema3A, VEGF165, NRP1 and CD3 expression in OA and RA synovial tissues
Expression of Sema3A, VEGF-A and NRP1 mRNA and correlation with DAS28-CRP in RA
Correlation of Sema3A mRNA levels with histological features in RA synovial tissues
Expression of NRP1 in CD20-positive B cells in lymphocyte aggregates
It has been suggested that abnormalities of the immune system play an important role in the pathogenesis of chronic inflammatory conditions such as RA. This study revealed Sema3A expression levels significantly correlated with the histological score of perivascular infiltrates of lymphocytes, focal aggregates of lymphocytes and diffuse infiltrates of lymphocytes. Sema3A is involved in immune responses, and its expression levels are altered in several autoimmune diseases [29, 37, 38]. The NRP1 and PlexinA complex mediates Sema3A signaling in the immune system . NRP1 is expressed by regulatory T cells, a subset of T cells  and PlexinA4 regulates T cell proliferation and activation through inhibition of the actin-cytoskeleton rearrangement and T cell receptor polarization . Recently, it was reported that Sema3A promotes regulatory T cells by enhancing IL-10 production . Regulatory T cells play an important role in maintaining immunological self-tolerance by suppressing autoreactive T cells . Immunohistochemical staining in the current study revealed that Sema3A is expressed in the lining layer. Thus, in OA joints, secreted Sema3A from the lining layer may enhance regulatory T cell functions to suppress autoimmune responses in the sublining layer. In RA, the reduction of Sema3A may abrogate the functions of regulatory T cells, thus allowing the infiltration and focal aggregation of autoreactive lymphocytes in the sublining layer. Further investigation of the correlation between Sema3A and IL-10 expression levels may increase our understanding of their roles in RA.
Catalano reported synovial tissues derived from healthy controls, OA and RA patients exhibited no significant differences although the relative Sema3A expression was lowest in RA samples . This discrepancy might be explained by the difference in sampling numbers, which can influence statistical power, and/or by different qPCR methods used. We quantified Sema3A mRNA expression in a larger number of RA patients (n = 30) and OA patients (n = 23), whereas Catalano used relatively small numbers of RA (n = 10), OA (n = 10) patients and healthy controls (n = 5). For qPCR, we used the standard curve method for relative quantification, whereas Catalano used a comparative Ct method.
Sequential immunohistochemical staining revealed that B cells were observed mainly in the lymphoid follicles, consistent with a previous report . Here we observed that B cells in the follicles expressed NRP1 (Figure 4). Vadasz et al. recently suggested that Sema3A can modulate the autoimmune properties of B cells in SLE . Thus, Sema3A may exert similar functions in NRP1-positive B cells from RA synovial tissues. Vadasz et al. also reported Sema3A serum levels were significantly lower in SLE patients (p < 0.0001) and RA patients (p = 0.047) compared with healthy controls . Since Sema3A is a secreted soluble protein and can enter the systemic circulation [16, 42], lower Sema3A serum levels in RA patients may reflect its reduced expression in knee joints and/or other organs. Additional investigations to quantify the serum levels of Sema3A from OA and RA patients may contribute to further elucidation of pathogenesis of the disease.
It is unclear which factors regulate Sema3A expression in synovial tissues. However, Fukamachi et al. reported that the presence of calcium and histamine could modulate Sema3A expression levels in human keratinocytes and fibroblasts . Since histamine is associated with RA pathogenesis [44, 45], it may be responsible for the decreased Sema3A levels observed in RA synovial tissues.
We found that mRNA expression of VEGF-A was not significantly altered in OA and RA synovial tissues. Hashimoto et al. reported staining intensity for VEGF expression did not differ between RA and OA synovial lining  and Lowin et al. reported that VEGF165 expression did not differ in the chronically inflamed tissue of RA patients and OA patients . In contrast, several reports showed altered expression of VEGF in RA. Lee et al. observed significantly higher levels of VEGF protein in RA compared with OA synovial fluid and serum . Kurosawa et al. observed significant correlations of serum VEGF levels with DAS28-CRP scores . The elevation of VEGF protein in RA synovial fluid and serum may explain the higher total number of VEGF-producing cells in the region . Indeed, our study showed no signification correlation between VEGF-A expression levels and DAS28-CRP scores (Figure 2), but there was a marked increase of synovial tissue thickness of the lining layer in RA (Figure 1). Thus, VEGF levels in RA serum may be increased as previously reported . In an earlier study, Ikeda et al. reported VEGF 165 was expressed in 41% of RA samples (17 patients) but not in OA samples (8 patients) using reverse transcription-PCR . They also found NRP1 was up-regulated in RA synovial tissues. However, we did not observe significant alterations of VEGF-A (Figure 2B) or NRP1 (Figure 2C) between OA and RA specimens. Kim et al. reported that NRP1 expression was similar in OA and RA using immunohistochemical analyses . These discrepancies might be explained by the difference in sampling numbers, disease duration of the populations studied, extent of inflammation, use of anti-rheumatoid drugs, and/or by different methods for analysis of NRP1 expression levels. Additional investigations in a large sample considering several different conditions are required to clarify these discrepancies.
Although Sema3A inhibits endothelial formation and angiogenesis due to competition with VEGF165 , this study did not identify a significant correlation between Sema3A mRNA expression levels and blood vessel density in RA synovial tissues. This suggests that angiogenesis in RA lesions may be regulated by other mediators, such as placenta growth factor 1, IL-2 and hepatocyte growth factor [47, 52].
The imbalance between Sema3A and VEGF may also affect the etiology of RA. Although VEGF-A expression levels did not exhibit a significant correlation with DAS28-CRP scores, the Sema3A/VEGF-A mRNA ratios demonstrated a relationship with the RA clinical score. Several studies have demonstrated that anti-NRP1 peptides suppressed the survival, adhesion and migration of VEGF165-induced synovial cells, which contribute to cartilage destruction in RA [50, 53]. VEGF165 also increased the production of cytokines by human peripheral blood mononuclear cells . Sema3A may inhibit the action of VEGF165 on synovial cells and inflammatory cells in a competitive manner.
Innervation is important in the pathogenesis of arthritis. Primary afferent sensory nerve fibers are proinflammatory, whereas sympathetic nerve fibers are anti-inflammatory . In RA synovial tissues, numbers of sympathetic nerve fibers decreased while sensory nerves increased when compared with OA . The loss of sympathetic nerve fibers in RA may be caused by increased Sema3C and its soluble receptor NRP2 [6, 55]. However, the mechanism of increased sensory nerve fibers in RA synovial tissues is not fully understood. Decreased Sema3A expression in RA may facilitate the increase of sensory nerves over sympathetic nerves in inflamed RA synovium .
Several limitations of our study should be addressed. First, this study population may be influenced by environmental factors as well as genetic factors. Second, patients enrolled in our study were in the advanced stages of RA disease (mean duration 16.3 years). Thus, our data do not reflect the pathogenesis of RA during the early stages. Third, anti-rheumatoid drugs may alter the expression level of Sema3A and other molecules. In this study 12 patients (40%) received methotrexate (MTX) (mean prednisolone daily dose 2.9 mg), 4 patients (13.3%) received TNF-α blockade (3 patients in combination with MTX, 1 patient in combination with sulfasalazine, mean prednisolone daily dose 3.6 mg), 11 patients received Disease Modifying Antirheumatic Drugs (DMARDs) without MTX (mean prednisolone daily dose 1.6 mg), and 3 patients received prednisolone alone (daily dose 6.2 mg). Although there was no statistically significant difference in Sema3A expression, DAS28-CRP, or Rooney’s inflammation score between the MTX–treated group and other groups (data not shown), we cannot rule out the possibility of altered Sema3A expression by anti-rheumatoid drugs, because each group sample size is small in this study.
This study demonstrated that Sema3A expression levels and the disease activity score in patients with RA negatively correlated with histological parameters in RA synovial tissues. This suggests that Sema3A may have a suppressive effect on the pathological severity of RA. Further elucidation of the role of Sema3A in joints may elucidate the biological and pathological functions of Sema3A and allow the development of new strategies for the treatment of RA.
28-joint disease activity score based on c-reactive protein
Hematoxylin and eosin
Quantitative real-time polymerase chain reaction
Systemic lupus erythematosus
Tumor necrosis factor-α
- VEGF165 :
Vascular endothelial growth factor 165.
The authors wish to thank Ms. Tamiyo Taniguchi, Mrs. Atsuko Fukui and Mrs. Inada Mae for their technical and secretarial assistance.
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